1. Field of the Invention
The present invention is related to the use of polarized light to visualize structures that possess molecular order or that are under strain.
2. Description of the Related Art
Materials having a different optical index of refraction for different states of polarization are said to express birefringence, and the amount of birefringence in a sample is termed its optical retardance. The index of refraction is highest for rays of light having an E field along a first direction, which is termed the fast axis for that sample. It is conventional to describe that direction as the azimuth angle, relative to some coordinate system of interest.
Polarized light has been used to obtain contrast in light microscopy. One benefit of this arrangement is that it enables one to obtain contrast with unstained samples. Common arrangements include use of a pair of crossed polarizers in the beam path, with one polarizer placed prior to the sample and one after it. The sensitivity of these methods is limited, and it is difficult to detect retardance is below 5 nm.
The Poincare sphere is an established way of representing state of polarization, where each point on the sphere indicates a unique polarization state of light. The longitude 2θ and latitude 2ε of a point on the sphere correspond to a polarization ellipse with azimuth θ and ellipticity angle ε. The ellipticity angle is an auxiliary angle that specifies the shape of the vibration ellipse, via the equation tan ε=b/a, where a and b are the major and minor semi-axes of the ellipse. Thus, lines of constant longitude and latitude on the sphere represent contours of equal azimuth and equal ellipticity, respectively. The Northern hemisphere represents light with right-hand elliptical polarization, and the Southern hemisphere represents left-hand elliptically polarized light.
In U.S. Pat. No. 5,521,705, Oldenbourg and Mai teach apparatus for calculating the optical retardance and azimuth angle at many locations in an unstained birefringent sample, and produce images of the sample based on this information. The apparatus illuminates a sample with quasi-monochromatic light that is approximately circularly polarized, and measures the intensity of light after it passes through an approximately circular analyzer polarizer. From four measurements of intensity, using slightly different polarization states at the illuminator polarizer or the analyzer polarizer, the birefringence properties of the sample are calculated and an image is displayed. One of the four measurements is taken with the two polarizers configured to produce substantially the best extinction possible; this configuration is termed the extinction state.
In U.S. Pat. No. 7,202,950 and U.S. Pat. No. 7,239,388, Oldenbourg and Shribak teach techniques for obtaining optical retardance and azimuth angle based on 2, or 3, or 5 measurements of a birefringent sample. They also teach the use of four measurements, where none of the states corresponds to the extinction state.
Cambridge Research and Instrumentation, Inc (Woburn, Mass.) manufactured the SpindleView and LcPolScope systems using the Oldenbourg and Mai technique, using a video camera and a personal computer to generate images of samples viewed in a microscope. Two liquid crystal cells and a linear polarizer are used to construct either the entrance polarizer or the analyzer polarizer. One of the liquid crystal cells provides approximately ½ wave of retardance, and the other provides approximately ¼ wave of retardance. The azimuth angles of the two cells are offset from one another by 45 degrees. An interference filter transmits a 30 nm band centered at 546 nm through the apparatus.
The software performs a calibration cycle wherein the video camera output is digitized and measured while the liquid crystal cells are driven to a variety of states. Based on the readings obtained under various trial conditions explored in this way, the software determines what are suitable settings for the liquid crystal cells. This calibration takes 30-60 seconds and must be performed before any of the normal operating functions are available. Once calibration is performed, these systems require approximately 2 seconds to acquire the video images, calculate an image of a sample, and produce an image on a computer display.
The SpindleView software has a button which engages or disengages a blinking mode. Calibration must have been performed beforehand. In blinking mode, the computer drives the liquid-crystal based polarizer between two states in alternation, which produces a blinking view at the microscope eyepieces. The blink rate is adjustable from about 1 state/second to a maximum of approximately 2.6 states/second via a software slider control. An observer looking through the microscope eyepieces sees the entire field of view change brightness, due to the changing configuration of polarizers. The view is not uniform: one sees markedly darker and lighter regions, and as the polarizer switches state, regions that were brighter may become darker, or their brightness may be unchanged. Also, while the image has an overall green appearance, there can be changes in hue, ranging from blue-green to yellow-green as different components of the imperfectly-pure green light vary in proportion. The details of what patterns are seen, and how they change during blinking, are unpredictable.
Nonetheless, within this blinking field, a skilled observer can learn to detect birefringent structures by their different blink signature: as the polarizer state is alternated, birefringent structures exhibit a greater or lesser degree of brightness (or color) change than do their surroundings. This is an unsatisfactory arrangement for several reasons: it is visually tiring due to the stroboscopic blink action; some people never attain competence at resolving structures against the background, or attain only limited competence so can only see the most highly birefringent structures; and the variable and uncontrolled nature of the spatial patterns make it hard to predict whether good results will be obtained in any given setup.
The Oosight system from Cambridge Research & Instrumentation (Woburn, Mass.) uses one or more of the techniques of Oldenbourg and Mai, or of Oldenbourg and Shribak, to produce computer-calculated images of birefringence in samples. The liquid crystal, polarizer optics, and interference filter are substantially the same as in the SpindleView system. This system has a live mode in which it takes approximately 3 images per second and displays calculated images to the computer display at this rate. It must perform a calibration step, similar to that in the SpindleView, prior to normal operation, during which the digitized images from the camera are measured by the computer software while the liquid crystal cell settings are adjusted.
The Abrio system from Cambridge Research & Instrumentation (Woburn Mass.) uses one or more of the techniques of Oldenbourg and Mai, or of Oldenbourg and Shribak, to produce computer-calculated images of birefringence in samples. The optics in this system differ from the Oosight, SpindleView and LcPolScope in that its entrance or analyzer polarizer incorporates three liquid crystal cells rather than two, together with a linear polarizer. Two of the cells are configured adjacent to one another with their azimuth angles offset by 90 degrees so they largely cancel one another, and produce a net retardance equal to the difference of their individual retardance values. Consequently, the arrangement has a retardance close to zero; the third liquid crystal cell has a retardance of ¼ wave and its azimuth angle is 45 degrees from either of the paired elements. Again, a narrow-band interference filter is used to produce monochromatic light from a source; either a 10 nm bandwidth or 30 nm bandwidth is used. It must perform a similar calibration step to that of the Oosight system.
These systems have been used in the fields of biology [Katoh 1999 Proc. Natl. Acad. Sci. USA. 96:7928-7931, LaFountain 2001 Mol Biol Cell 12:4054-4065], materials science [Hoyt 1999 American Laboratory. 31(14):34-42] and medicine [Keefe 2003 Reprod Biomed Online 7(1):24-9, Shen 2005 Human Reproduction].
Keefe reported that the Oldenbourg and Mai technique can be used to detect, locate, and evaluate the condition of organelles within oocytes. Shen has used it for quantitative assessment of these structures. In particular, a structure called the spindle can conveniently be detected in this way, though it is ordinarily invisible. The physical origin of the optical retardance in such structures arises from the fact that their molecular arrangement is ordered, which gives rise to birefringence.
In the field of somatic-cell nuclear transfer, where oocytes are enucleated to serve as hosts for DNA from an organism that is to be cloned, practitioners report using the Oldenbourg and Mai apparatus to assist with visualization of the spindle [Wang 2002 Cloning Stem Cells 4(3):269-76]. Mitalipov reported successful cloning of a monkey to produce stem cells, in a process where the Oosight apparatus was used as an aid during enucleation, to locate the spindle.
MTG Medical Technology (Altdort, Germany) sells the ICSI-Guard system for use in embryology and in vitro fertilization procedures. It includes a camera and image digitizer which takes images of a sample using polarized light, and produces a computer-generated image of structures in the sample on a display.
Schimming and Rink teaches apparatus for polarized light imaging in WO2006/081791.
Structures in unstained samples can be observed using other methods, such as differential interference contrast, phase contrast, interferometry, Nomarski contrast, and Hoffman contrast. However, each of these has strengths and weaknesses, and in general no one technique is effective in all cases. All are of limited value for observing spindle structures in oocytes.
The apparatus of the prior art provides for measurement of birefringent structures at rates ranging from several seconds per image, to approximately 3 images per second. It also provides for a visual detection of birefringent structures based on interpretation of a blinking eyepiece view of a sample by a skilled operator while polarizers are alternately driven between different states, at rates up to approximately 3 states/second. This art involves complicated and costly systems with a digitized camera and a personal computer; first to perform the initial calibration and setup; and then, to perform the measurement of intensity, calculate the sample retardance, and generate an image on a computer display. Where a quasi-real-time view is available, it is provided at a computer display so the microscopist must shift attention from the microscope to the display; or yet more complex systems must be devised which project or merge the computer-generated image into the eyepiece view. Where an eyepiece view is provided, it is of limited value for reasons such as those noted above, and the green-light view it affords is undesirable to some users.
Indeed, in the prior art for measuring low birefringence structures in samples, the use of quasi-monochromatic light is integral to the measurement. Yet this interferes with other uses of the microscope for several reasons. First, switching to any white-light mode requires at least the removal of the filter element, or the reconfiguration of the illuminator, so as to obtain a white-light view. Where this is done via a filter, the microscopist suddenly is presented with a much brighter view than before, since the entire illumination flux is now seen, rather than just a small portion of the green component. This can lead to uncomfortable, even painful, glare and a period of accommodation.
Also, the prior art methods have inherently low transmission, since they use polarizers operated near their extinction point. Consequently, microscopists choose a relatively bright setting for the microscope lamp, to provide an adequately bright view. Switching to a different microscopy mode such as bright field, Nomarski, or Hoffman (relief) contrast may require removing one or more of the polarizers from the beam. Without the polarizer extinction, the signal in the eyepieces becomes much brighter. When this is combined with the brightness increase from switching to white light from quasi-monochromatic green light, the signal is vastly different in the configuration where birefringent structures are visible, from that in other microscopy modes. Often the microscopist needs to adjust the lamp or introduce attenuators to obtain a satisfactory view. This sort of adjustment is in addition to the adjustment whereby the polarizer must be removed from the beam or reconfigured in some way. So changing between the birefringence imaging modes of the prior art, and other microscopy modes often involved several user adjustments and significant brightness adjustment or changes.
There are many areas of technical work where a microscopist uses multiple imaging modes. This is because certain tasks are more readily performed with a particular view of the specimen. For example, someone performing in vitro fertilization may prefer to use a prior art birefringence imaging method such as the Oosight to view the spindle, but prefer to use a different contrast technique such as Hoffman contrast for egg manipulation.
In the prior art, the two modes may not be employed simultaneously, for several reasons. First, there is a tremendous light loss if both systems are engaged. The prior art system of Oldenbourg uses circularly polarized quasi-monochromatic light, such as a 30 nm band in the green centered on 546 nm. Overall, less than 10% of the total visible light reaches the sample—all other wavelengths are discarded, as is light in the complementary polarization state. Then, light must pass through the analyzer polarizer, which is set to an operating point near extinction, so it transmits only 2%-5% of the light reaching it. Overall, the apparatus transmits 0.2-0.5% as much light as a simple transmitted light setup, and light levels are often marginal for this apparatus, especially when operated with high magnification objectives such as 20× or higher.
The Hoffman arrangement requires placing a patterned mask at the back focal-plane of the objective, and another at a conjugate plane on the illumination optics side; these further attenuate the light approximately 10-fold. Part of the loss is because numerical aperture is reduced, and part is because neutral-density elements or linear polarizers are cover some of the pattern area.
The combination of all these losses leads to an unusably dark image.
Second, some modalities use polarized light optics in ways that conflict with the polarization measurements of the prior art birefringence imaging systems. For example, some Hoffman implementations incorporate an adjustable linear polarizer on the illumination side, which works in concert with a linear polarizer covering one or more slots of the slotted mask. These form a variable attenuator, where the user rotates the adjustable linear polarizer to achieve an optimum sample view, based on the degree of attenuation and the image produced.
The prior art apparatus utilizes a circular polarizer on the illumination side, and teaches circular polarizers consisting of a linear polarizer followed by a quarter wave plate. The variable linear polarizer interacts with the linear polarizer within the circular polarizer in undesirable ways. For example, it is possible to achieve a setting where no light at all passes through the system because these two polarizers are crossed. Yet that setting of the variable linear polarizer may correspond to the optimum sample view setting for Hoffman imaging.